TW202146659A - Comparing copies of polynucleotides with different features - Google Patents

Abstract

Provided is a method including making copies of two or more populations of polynucleotides including identifier sequences, wherein the copies are attached to a substrate, hybridizing oligonucleotides to the identifier sequences, and comparing an amount of oligonucleotides hybridized to the copies of the two or more populations of polynucleotides, wherein at least one feature differs between the two or more populations of polynucleotides or between the making of the copies of the two or more populations of polynucleotides attached to the substrate.

Description

Compare copies of polynucleotides with different characteristics

This application relates to methods of making copies of a population of polynucleotides comprising an identifier sequence, hybridizing oligonucleotides to the identifier sequence, and detecting or comparing the amount of hybrid oligonucleotides.

Many current sequencing platforms use "sequencing by synthesis" (SBS) technology and fluorescence-based detection methods. In some examples, a number of polynucleotides isolated from one or more populations of nucleotides to be sequenced are attached to the surface of the substrate and copied. SBS can then be performed on the surface-linked copies. Making copies of a polynucleotide or amplifying a polynucleotide and sequencing the copies increases the fluorescent signal emitted during sequencing, and thereby enhances the sequencing process.

Copies of the polynucleotide attached to the substrate can be synthesized by solid-phase nucleic acid amplification methods that immobilize the amplification products on a solid support to form an array comprising clusters of immobilized nucleic acid molecules. Each cluster or colony on such an array is a plurality of copies of a target polynucleotide strand and a plurality of immobilized polynucleotide strands complementary thereto. Cluster amplification methods or clustering methods are examples of methods in which surface-ligated copies and complementary sequences of a polynucleotide of interest are synthesized for SBS. Some examples of suitable methods that may also be used to generate surface-linked copies, etc., include bridge amplification, kinetic exclusion amplification ("ExAmp"), or other methods.

Clustering involves the use of polymerases to synthesize surface-linked clusters. However, a known problem with certain polymerases and polymerization methods is the quantitative synthesis bias associated with various characteristics of the target polynucleotide. For example, in some cases, clustering methods may be biased toward amplifying more guanine (G)-cytosine (C) bases with lower percentages than polynucleotides with relatively higher GC content The base pair's copy of the target polynucleotide. In other cases, the clustering method may be biased towards amplifying more copies of relatively short polynucleotides of interest relative to relatively long polynucleotides. In still other examples, other theoretical sources of bias may affect the relative amplification levels of polynucleotides, such as polynucleotide sample preparation methods or other differences.

At least in view of the foregoing, sequencing techniques would therefore benefit from determining the presence of such biases during clustering and other amplification processes, and identifying, isolating and modifying such techniques could minimize such biases and yield more precise sequencing method of results.

In one aspect, there is provided a method comprising: making two or more copies of a population of polynucleotides comprising an identifier sequence, wherein the copies are attached to a substrate such that the oligonucleotides are associated with the identifiers Sequence hybridization, and comparing the amount of oligonucleotides hybridized to copies of the two or more polynucleotide populations, wherein at least one characteristic is between the two or more polynucleotide populations Or differ between the production of copies of the two or more polynucleotide populations attached to the substrate.

In one example, the at least one characteristic is selected from the group consisting of length, guanine-cytosine content, and method of preparation. In another example, the at least one characteristic includes guanine-cytosine content. In yet another example, the at least one characteristic includes length. In yet another example, the at least one feature includes a method of making. In another example, at least one characteristic differs between the production of copies of the two or more polynucleotide populations attached to the substrate. In yet another example the oligonucleotides comprise fluorophores.

In one example, the method further comprises detecting a difference between the amount of oligonucleotides hybridized to copies of the two or more polynucleotide populations attached to the substrate, wherein the difference is at least about 10%. In another example, the difference is at least about 20%. In yet another example, the difference is at least about 30%.

In one example, the at least one feature includes a combination, and the combination includes guanine-cytosine content, length, method of preparation, and production of copies of the two or more polynucleotide populations attached to the substrate two or more, the two or more polynucleotide populations include three or more polynucleotide populations, and each of the three or more polynucleotide populations The combination is different from the combination of another polynucleotide population.

Another example further includes detecting a difference between the amount of oligonucleotides hybridized to copies of two or more of the three or more polynucleotide populations attached to the substrate, wherein The difference is at least about 10%. In one example, the difference is at least about 20%. In another example, the difference is at least about 30%.

In another aspect, there is provided a method comprising: making two or more copies of a population of polynucleotides comprising an identifier sequence, wherein the copies are attached to a substrate such that an oligonucleotide comprising a fluorophore The acid hybridizes to the identifier sequences, and detects the amount of oligonucleotides hybridized to copies of the two or more polynucleotide populations, wherein at least one characteristic is in the two or more Differences between populations of polynucleotides or between the production of copies of the two or more populations of polynucleotides attached to the substrate, and the at least one characteristic is selected from the group consisting of length, guanine-cytosine content , methods of preparation and manufacture of copies of the two or more polynucleotide populations linked to the substrate.

In one example, the at least one characteristic includes guanine-cytosine content. In another example, the at least one characteristic includes length. In yet another example, the at least one feature includes a method of making. In yet another example, at least one characteristic differs between the production of copies of the two or more polynucleotide populations attached to the substrate.

Another example further includes detecting a difference between the amounts of oligonucleotides hybridized to copies of the two or more polynucleotide populations, wherein the difference is at least about 10%. In one example, the difference is at least about 20%. In yet another example, the difference is at least about 30%.

The present invention relates to a method for assessing bias in polynucleotide copies such as those that are part of the SBS process. In particular, includes a process of identifying the presence of a bias for relatively more or less copies of a polynucleotide made by a given population relative to copies made by a different population. Polynucleotides of different populations can be distinguished from one another by characteristics that differ from one population to another. The characteristic can be any characteristic of the polynucleotides of the population, including the physical properties of the polynucleotide strands or the processes that the polynucleotide population undergoes as an aspect of sample preparation.

For example, a polynucleotide from one population can have a lower or higher ratio of C and/or G bases:A and/or T bases relative to a polynucleotide from another population. In another example, polynucleotides from a population can be several nucleotides long, wherein the length of the polynucleotides of one population is different from the length of the polynucleotides of the other population. In another example, different polynucleotide populations may have undergone different production methods. For example, it may have undergone different methods of fragmenting target molecules into shorter polynucleotides for copying and sequencing, or adding oligonucleotide sequences or identifiers to polynucleotides (with a process known as indexing, indexing, or barcoding, which is the way in which a polynucleotide is labeled or indexed to identify subsequently manufactured copies of it), or the association of a polynucleotide sequence with an original sample Different methods of separation (such as separation of polynucleotides that select a predetermined size or range of predetermined sizes). In other examples, the method of forming clusters from one population of polynucleotides can be different from the method of forming clusters from different populations of polynucleotides.

In some instances, any characteristic, whether directly related to a physical characteristic of a polynucleotide of a different population, or indirectly indicative of a characteristic of the polynucleotide itself as a result of its preparation, storage, handling, manipulation, or process of preparation or clustering, or is associated with Correlation of other characteristics, such as other components that may be present with the polynucleotide, can distinguish two or more populations. The methods as disclosed herein can be used to determine whether differences in features are causing a bias in copying, such as during a clustering process, that results in a disproportionately higher amount of polynucleotides being copied by one population relative to another population or rate.

In some examples, populations may differ in more than one characteristic, including characteristics from GC content, length, method of preparation, or a characteristic in a process such as making copies during clustering. For example, the length of a population (eg, the number of nucleotides in a population polynucleotide) and GC content (eg, G and/or G in a polynucleotide population compared to A and/or T residues in a polynucleotide population) and/or relative amounts of C residues) may vary. Or it may differ with respect to any of these and methods of preparation, methods of copying during clustering, or any combination of two or more of the foregoing. In some examples, a population can be related to one or more characteristics or to a combination of any two or more characteristics, or to a combination of any three or more characteristics (such as length, GC content, or polynucleotide production) by the method used for copying or clustering, and or copying methods such as clustering methods).

Variations in the methods of making different populations of polynucleotides that characterize a population can confer different structural characteristics on the population, such as the effectiveness of obtaining polynucleotides of a desired size, the uniformity of polynucleotide size within a population, how many within a population Polynucleotides suitably have differences in adapters or other sequences linked thereto, etc., which can all cause biases or copy differences that manifest after clustering. The methods disclosed herein can be used to determine such effects of differences in manufacturing methods.

In some examples, the characteristics of one or more of the two or more polynucleotide populations may be preselected, including any of the foregoing characteristics or any two or more of the foregoing characteristics in combination with each other. For example, it may be beneficial to determine whether other aspects of the clustering process or copying process cause, increase, decrease, eliminate, or otherwise affect polynucleotide length, GC content, production process, or other characteristics, or the rate at which copies are made. method, or any combination of two or more of them. Thus, the characteristics of a polynucleotide population can be preselected and configured to reflect such potential or hypothesized causes or sources of bias, as well as the clustering or other copying processes performed and the two or more polynuclei being compared The amount of copies of the nucleotide population. By normalizing the starting amount of each at the onset of copying, the amount of copies in one population that is greater than the amount of copies in another population can be indicative of a polynucleotide with a preselected characteristic for or against the copy conditions used. the bias.

An example of this method is illustrated in the flowchart of FIG. 1 . Two or more polynucleotide populations are prepared for copying, such as by a clustering process. The preparation process involves adding an oligonucleotide sequence to a polynucleotide of a population and adding another oligonucleotide sequence to a polynucleotide of another population. In instances where more than two populations of polynucleotides are used, oligonucleotide sequences may be added to the polynucleotides of the population that differ from the polynuclei added to each of the other populations An oligonucleotide sequence in nucleotides such that the polynucleotides of each population include oligonucleotides that are specific to the polynucleotides of that population and are different from oligonucleotides added to the polynucleotides of any other population acid sequence. Differences in the sequence of such oligonucleotides, referred to as identifier sequences, allow them to hybridize to oligonucleotides having sequences complementary thereto. For example, each identifier sequence added to a polynucleotide of two or more of these populations can hybridize to an oligonucleotide sequence that is any of its two or more populations The identifier sequences of the other polynucleotides are not hybridizable. As set forth further below, the presence of an identifier sequence whereby polynucleotides from different populations can be distinguished may allow for the identification of copies of polynucleotides from a given population, but not any other, according to the methods as disclosed herein group.

Two or more populations of single-stranded polynucleotides can then be copied, where the copies are attached to the substrate. For example, as mentioned above, copying may be performed according to a solid state exclusion amplification clustering process, bridge amplification clustering, or other processes. In a non-limiting example, the 3' end of the polynucleotide can be hybridized to a primer sequence attached to the substrate, and a polymerization process performed to generate the complementary sequence of the polynucleotide, starting from the surface-attached primer and extending to each polynucleotide The complementary sequence to the 5' end of the acid. Polynucleotides from two or more populations can then be amplified from their surface ligated complementary sequences. According to a bridge PCR procedure, as a non-limiting example, the free 3' ends of the surface-linked complementary sequences of the polynucleotides of the two or more populations can then be hybridized to another primer sequence linked to the substrate. The complementary sequence can then be copied by a polymerase reaction, producing copies of the polynucleotides of the two or more polynucleotide populations, and their complementary sequences extending from the surface. The surface-bound complementary sequence and the copy can then be dehybridized from each other, and another round of polymerization is performed in which the surface-bound copy of the polynucleotide of the two or more polynucleotide populations and its complementary sequence (connected to its free 3) are copied. After the 'end is hybridized to the surface-linked primer, which serves as the initiation site for the polymerase reaction), the complementary pairs of surface-linked polynucleotides are subsequently dehybridized from each other. By repeating this process, clusters of copies of the polynucleotides and their complements of two or more populations linked to the substrate can be formed. Other comparable methods of making copies of a polynucleotide population can also be used in other examples, whether PCR, rolling circle amplification, multiple displacement amplification, random priming amplification, isothermal amplification, and the like.

The amount of substrate-attached copies of a polynucleotide from one of the two or more polynucleotide populations can then be determined. For example, an oligonucleotide that can hybridize to an identifier sequence of a polynucleotide of one of two or more populations can be added such that it matches the identifier sequence present on the polynucleotides mixed. Hybridizable oligonucleotides can include detectable labels, such as fluorescent labels capable of emitting detectable fluorescence upon stimulation by electromagnetic radiation of a given wavelength. By inducing such hybrid oligonucleotides to fluoresce and detecting the amount of fluorescence emitted, the amount of copies of the polynucleotide from one of the two or more populations can be assessed.

The oligonucleotide can then be dehybridized and then incubated with another oligonucleotide that can be combined with a polynucleotide of the other of the two or more polynucleotide populations The identifier sequence is heterozygous. The other hybridizable oligonucleotide may include a detectable label, such as a fluorescent label capable of emitting detectable fluorescence upon stimulation by electromagnetic radiation of a given wavelength. By inducing such other hybrid oligonucleotides to fluoresce and detecting the amount of fluorescence emitted, the amount of copies of the polynucleotide from the other of the two or more populations can be assessed. In instances where potential copy bias is caused or arises from features or combinations of features of polynucleotides of more than two polynucleotide populations, each identifier sequence of polynucleotides of individual nucleotide populations may be complexed with each other Hybrid oligonucleotides, the process of measuring the amount of hybrid oligonucleotides, followed by its de-hybridization (if there is subsequent hybridization of another oligonucleotide), to obtain the results of various types of oligonucleotides. A measure of quantity as a measure of the quantity of copies of a polynucleotide from each of two or more populations of polynucleotides.

In one example, the difference between the amounts of oligonucleotides that are hybridized to copies of each of the two or more polynucleotide populations can be, for example, hybridized to the respective identifier sequences by comparison. detected by the relative amount of fluorescence emitted by the oligonucleotide. For example, a sample can include two populations of polynucleotides that include different identifier sequences and define features by having different characteristics. Different samples can include different relative proportions of polynucleotides from each of the two populations. For example, a population can constitute about 0%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40% of the total nucleotide content of the sample %, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 100%, and another population Make up the rest of the sample. Copies of the population and its complement can then be made in accordance with the disclosure herein, such as in a clustering process.

The oligonucleotides can then hybridize to an identifier sequence of copies of the polynucleotide population. The amount of hybrid oligonucleotides can be measured, such as in one instance where the oligonucleotides include a fluorophore, and fluorescent emission can be detected and quantified as oligonuclei hybridized to an identifier sequence of a given population A measure of the total amount of nucleotides. In this way, the amount of oligonucleotides heterozygous with each population can be measured and compared, resulting in an indication of the relative abundance of copies of the polynucleotides of each population after copying. In one example, differences may be detectable when the samples include different relative proportions of each polynucleotide population. For example, when a population constitutes about 0%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30% of the nucleotide content of a sample prior to copying, such as by clustering %, about 35%, about 40%, or about 45%, and another population makes up the remainder, the difference may be detectable.

In one example, fluorescence emission is measured from oligonucleotides comprising a fluorophore hybridized to the identifier sequence of each population, and differences in fluorescence are determined. For example, an oligonucleotide that can hybridize to an identifier sequence of one population of polynucleotides can include a detectably different oligonucleotide that can hybridize to an identifier sequence of another population of oligonucleotides fluorophores such that fluorescence emission from one can be detected independently of fluorescence emission from the other, and vice versa (eg, Alexa 647, Alexa 532, etc.). In one example, the fluorescence emitted by an oligonucleotide hybridized to one identifier sequence may be at least about 10% greater or less than the fluorescence emitted by an oligonucleotide hybridized to another identifier sequence, or greater or lesser At least about 15%, or at least about 20% larger or smaller, or at least about 25% larger or smaller, or at least about 30% larger or smaller, or at least about 35% larger or smaller, or at least about 40% larger or smaller, At least about 45% larger or smaller, or at least about 50% larger or smaller. In another example, an oligonucleotide hybridized to one identifier sequence may emit about 10% greater or less fluorescence than an oligonucleotide hybridized to another identifier sequence, or greater or lesser About 15%, more or less about 20%, more or less about 25%, more or less about 30%, more or less about 35%, more or less about 40%, more or less about 45% %, more or less about 50%.

An example is shown in FIG. 2 . In the far left panel area, two polynucleotides are shown, one from each of the two polynucleotide populations. Each includes an index or sequence of identifiers. In practical examples, a plurality of polynucleotides from each of two or more populations can be used. Show the surface on which solid phase copying will occur. In this example, the surface is the surface of the fluid bath. Primers attached to the surface (eg, primers P5 and P7) are complementary to and can hybridize to a portion of the 3' end of the polynucleotide. The polynucleotide is then hybridized with surface-linked primers, which are then extended by a polymerase to form the complementary sequence of the polynucleotide. The polynucleotide is then dehybridized, leaving a surface-linked complementary sequence, extending from what was originally a surface-linked primer. Polymerization using the surface-linked complementary sequences of the polynucleotides of two or more populations as templates results in the formation of surface-linked copies of the polynucleotides of the populations. Such strands are then linearized and dehybridized from each other, after which the process is repeated. By iteratively repeating this process, the number of surface-linked copies of the polynucleotides of two or more populations and their surface-linked complements are amplified, resulting in surface-linked clusters. A set of strands representing copies of a population of polynucleotides or complementary sequences can then be removed from the substrate, such as by enzymatic cleavage. See the arrows in Figure 2 indicating "Clustering and Linearization".

If characteristics or combinations of characteristics that distinguish one from the other of two or more populations cause a bias in the copying process, or if the copying process affects differently, such bias may be reflected in the In the difference between the amounts of surface-linked copies of a polynucleotide of a population. Such differences can be achieved by hybridizing oligonucleotide probes that can hybridize to an identifier sequence of one population but not to any other population and carry a detectable linker, such as a fluorescent label to make sure. As shown in the third panel of Figure 2, such probes can hybridize to copies of a population, wash away excess unbound probe, and subsequently induce induction from oligonucleotides linked to oligonucleotides by measuring The amount of hybrid probe is detected by how much fluorescence is emitted after stimulating the surface with electromagnetic radiation of a certain wavelength emitted by the fluorescent label of the acid probe.

Subsequently, the first probe can be dehybridized and washed away, followed by hybridization with another probe. This other oligonucleotide can hybridize to an identifier sequence of another population (but not to any other population's identifier sequence) and carry a detectable linker, such as a fluorescent label. As shown in the last panel of Figure 2, this other probe can hybridize to a copy of this other population, wash away excess unbound probe, and subsequently induce induction from ligation in the The amount of hybrid probe is detected by how much fluorescence is emitted after the surface is stimulated with electromagnetic radiation of a certain wavelength emitted by the fluorescent label of the other oligonucleotide probe. Comparing how much fluorescence is detected from the first hybrid probe and from the second hybrid probe indicates the difference in how many copies of the polynucleotide from both of the two or more populations are attached to the surface.

By comparing this difference to the difference in the amount of polynucleotides from each of the two or more polynucleotide populations used to initiate the clustering process, one or more can be identified that distinguish two Effects of characteristics of one or more populations of polynucleotides on copy bias or bias generated by the method of copying. That is, by comparing the amount of each such oligonucleotide that hybridizes to copies of each of the two or more populations with each other, it is determined by the amount of polynucleotides from each population used for copying. Relative quantitative normalization can determine the presence and magnitude of a given copy bias. For example, if a feature causes a bias in copying, it is from two of the features defined by that feature (such as higher GC content, longer length, sample preparation process, any combination of two or more of the foregoing, etc.) The relative amount of copies of a polynucleotide of one of the one or more populations of polynucleotides may exceed that from a characteristic that is differently defined by that characteristic (such as lower GC content, shorter length, different sample preparation procedures, the aforementioned the relative amount of copies of the polynucleotide of the other of the two or more polynucleotide populations, another different combination of two or more of them, etc.). In turn, detection of such differences can indicate a bias toward or against copies of the polynucleotide characterized by the feature or combination of features.

The solid state amplification process results in the formation of copies and complementary sequences of polynucleotides from the initial population bound to the surface. A copy of the polynucleotide includes the identifier sequence. In turn, the complement of such copies includes the complement of the identifier sequence, and the complement of the identifier sequence may also be uniquely hybridized to not hybridized to other copies or complements of the polynucleotide attached to the surface. Oligonucleotide probes. The oligonucleotides are hybridized to the surface of the polynucleotide to bind an identifier sequence on the copies and the amount of such hybrid oligonucleotides is measured, which indicates how many copies of the polynucleotide occurred during the copying process. Similarly, oligonucleotides are hybridized to the surface of a copy of a polynucleotide that binds the complement of an identifier sequence on a complementary sequence and the number of such hybrid oligonucleotides is measured, which is also indicative of during the copying process How many polynucleotide copies occur. The amount of probes that detect the complement of an identifier sequence that can hybridize and hybridize to a surface-linked copy of a polynucleotide, or a surface-linked identifier sequence on a complementary sequence, can be used as a measure of how many copies of a population of polynucleotides occur. instruct.

Although in some instances no polynucleotides from two populations may have identifier sequences identical to each other, in other instances polynucleotides from two or more populations may include identical identifier sequences to each other . For example, a polynucleotide can include more than one identifier sequence. Polynucleotides from all two or more populations of polynucleotides can have a first identifier sequence unique to each population. It may have a second identifier sequence that is common between two or more of the two or more populations but different from any other of the two or more populations. A population may have a third sequence of identifiers that is also shared by some populations but distinguished by others. The population may further have a fourth identifier sequence common to all populations. In this example, differences between populations for a given identifier sequence can be such that an oligonucleotide that can hybridize to one sequence at such a hybrid sequence cannot hybridize to another sequence at such an identifier sequence. The population of polynucleotides from which surface-bound copies and complementary sequences are derived can thus be determined by hybridizing probes specific for a given identifier sequence.

In a non-limiting example, there may be four populations of polynucleotides. Two populations can have higher GC content than the other two populations, and two populations can have polynucleotides that are longer in length than the other two populations. Length and GC content can be mixed between four populations, where the first population has long polynucleotides with high GC content, the second population has long polynucleotides with low GC content, and the third population has short polynucleotides with high GC content nucleotides, and the fourth population has short polynucleotides with low GC content. Each population can have one, two, three, four or more identifier sequences. The first identifier sequence may be unique to each population. The second identifier sequence is distinguishable between populations of different lengths, wherein the first and second populations have the same second identifier sequence as each other and the third and fourth populations have the same identifier sequence as each other, wherein The second identifier sequences of the first population and the second population are different from the second identifier sequences of the third population and the fourth population. The third identifier sequence is distinguishable between populations with different GC content, wherein the first and third populations have the same second identifier sequence as each other and the second and fourth populations have the same identifier sequence as each other, The third identifier sequences of the first group and the third group are different from the third identifier sequences of the second group and the fourth group. The fourth identifier sequence can be shared by all groups.

Following copying, the heterozygosity of probes specific for the sequence of a given identifier sequence, and the measure of their heterozygosity, can be indicative of different populations or differential combinations of polynucleotides according to the characteristics that distinguish them or share them, Different amounts of surface bound copies, ie how many copies occur. For example, the amount of each population can be individually determined by measuring hybridization of probes specific for each sequence of the first hybrid sequence. The copy amounts of the long and short polynucleotides can be determined by measuring the hybridization of the oligonucleotide to each sequence of the second identifier sequence. The copy amounts of high GC and low GC content polynucleotides can be determined by measuring the hybridization of the nucleotides to each sequence of the third identifier sequence. And the total copy amount can be determined by measuring the hybridization of nucleotides to the fourth identifier sequence. In other examples, a greater or lesser number of identifier sequences may be included in some or all polynucleotide populations, and combined in different ways in different populations. In other instances, there may be more than two sequences that may be present in a given identifier sequence, such as several instances in which a given feature is compared (eg, low, medium, or high GC content, or short, medium, and long polynucleotides length, etc.).

After clustering but before hybridization of the oligonucleotides to the identifier sequence, copies of the polynucleotides of the two or more populations and complementary sequences are bound to the surface as described above. It may be advantageous to remove the surface-bound complementary sequence prior to assessing hybridization of the oligonucleotide to the identifier sequence. Alternatively, in another example, it may be advantageous to remove the surface-bound copy prior to measuring hybridization of the oligonucleotide to the complement of the identifier sequence on the surface-bound complement. Removing surface-bound copies or complementary sequences of polynucleotides of two or more populations can be accomplished by including selectively cleavable residues in the surface-bound primers from which such copies and complementary sequences extend, The copies or complementary sequences extending therefrom are thus removed after clustering. For example, a primer can include a deoxyuridine (dU) moiety. Subsequent treatment with an enzymatic formulation, such as LMX1, can cleave the primer at the dU residue and release the polynucleotide extending therefrom. In another example, the surface-attached primer can include an 8-oxoguanine (penox-G) residue. Subsequent treatment with an enzyme formulation, such as LMX2, can cleave the primer at the pendant oxy-G residue and release the polynucleotide extending therefrom.

In addition, aspects of copying processes, such as clustering processes, can be modified or compared to determine whether such aspects are reduced, eliminated, partially eliminated, exacerbated, or otherwise affected by biases resulting from the characteristics of a polynucleotide population. For example, copying can be performed under different conditions if a feature that distinguishes the polynucleotide from two or more different populations is generated, causes the feature, or is determined to be associated with the feature according to methods as disclosed herein, Processes such as clustering, and the effect of such differences in copying conditions on such biases can be determined. The aspect of the process of making copies of a polynucleotide population (eg, the aspect of the clustering process) can be a feature, and such features can vary from population to population. In one example, polynucleotides from two different populations that differ in a first characteristic (such as, by way of non-limiting example, GC content, length, and/or preparation process) can be used in two different conditions (a second characteristic) Copy of each of them. Any difference in the amount of copies of the polynucleotides from the two populations that represents the first characteristic associated with the bias of the copies when copied under one set of conditions can be subsequently compared to when copied under another set of conditions from the two Any differences in the number of copies of the polynucleotides of the populations are compared. If such differences were different from each other, it would indicate that differences in copy conditions due to a bias associated with a trait may be affected by the condition (ie, possibly by a second trait). In another example, a distinguishing characteristic may be an aspect of the method of making copies of two or more polynucleotide populations, such as an aspect of a clustering process, and no other characteristic that is different.

In one example, the characteristic correlation bias reflected in the difference between the copy numbers of the two populations when copied under one set of conditions is comparable to the difference between the copy numbers of the two populations when copied under another set of conditions. The traits reflected were less or more biased (indicated by smaller or larger differences in the number of copies between the two populations). Any component, situation, environment, or other aspect of the copying can be modified or tested for effect on the bias reflected in the difference in copy number between the two polynucleotide populations for the distinction of characteristics. For example, different polymerases, additives in the polymerization reaction (such as polyethylene glycols, salts, nucleotides, etc.), substrates, polymer coatings of substrates, fluid cell characteristics, temperature, timing or number of polymerization cycles, Components for rehybridizing or linearizing copies of polynucleotides and their complements (eg, LMX1 or LMX2, after clustering, but before resynthesis of surface-linked polynucleotides for subsequent sequencing rounds) used in some biochemical processes to release a subset of surface-linked polynucleotides) or any other conditions can be modified and compared. More than one instance of the condition is comparable to more than one other instance. In addition, a number of conditions can be modified to determine whether there is an interaction therebetween, eg, on a feature-related copy bias. In addition, multiple features can be compared for their individual and combined effects on bias as described above, and one or more conditions (alone, in combination, or both) can be compared with respect to bias associated with any one or more features impact to be tested.

Methods according to the present invention provide advantages over other methods for assessing potential bias in clustering or other copying processes for use in next-generation sequencing technologies. For example, as disclosed herein, the source of bias can be assessed without sequencing the copied polynucleotide. Potential sources of bias and possible adjustments to minimize, eliminate, or otherwise affect bias can be identified without the additional time, expense, and computational burden of performing and analyzing polynucleotide sequencing. Furthermore, the examples disclosed herein provide high-throughput methods for assessing multiple sources of potential bias, such as polynucleotide signatures, either individually or in combination, and modifications of which may result in the elimination or otherwise modification of A number of variables of bias, such as the conditions under which or according to which copies occur, eg, clustering or otherwise conditions that may affect any aspect of polynucleotide copying that occurs prior to sequencing in the SBS process.

With regard to assessing the bias attributable to GC content as a feature, a polynucleotide population can be characterized by an average relative GC content. For example, certain microbial species are known to have relatively higher or lower average percentages of GC content compared to, for example, humans, who on average have approximately equal proportions of GC and AT content. Some microorganisms, such as bacteria of the Rhodobacter group, are known to have higher GC content, such as above 60% GC content. Other microorganisms, such as Bacillus cereus are known to have lower GC content, such as below 40% GC content. In one example, the GC content can be characteristic. A population of polynucleotides may be polynucleotides prepared from Rhodobacter that are characterized by higher GC content relative to other populations, another population prepared by humans and characterized by moderate GC content relative to other populations, or Prepared from Bacillus cacti, which is characterized by a lower GC content relative to other populations. "Higher" and "lower" are used relatively here. Thus, using humans as a population, depending on the GC content of another population (eg, Bacillus cacti and Rhodobacter, respectively), it may be characterized by a higher GC or a lower GC relative to this other population.

In other examples, polynucleotides can be derived from synthetic or artificial sources with predetermined GC content by, for example, directly determining the sequence of a polynucleotide in a sample or the relative relationship of nucleotides of a given type in a stoichiometric control strand The amount of incorporation depends on the method of its synthesis (eg, using template-independent methods for sequence synthesis). A polynucleotide population can include any desired or known percentage of GC, meaning the total combined number of guanine and cytosine nucleobases in the total number of nucleobases (G, C, A, and T total). A population can be defined by the GC content that is characteristic of the population as a whole, even though individual polynucleotides of the population can have a different GC content than the GC content of the population as a whole.

The population can have about 5% GC content, about 10% GC content, about 15% GC content, about 20% GC content, about 25% GC content, about 30% GC content, about 35% GC content, about 40% GC content , about 45% GC content, about 50% GC content, about 55% GC content, about 60% GC content, about 65% GC content, about 70% GC content, about 75% GC content, about 80% GC content, about 85% GC content or about 90% GC content, or any intermediate amount of GC content. All other possible comparisons are expressly included as aspects of the invention.

With regard to assessing bias attributable to polynucleotide length, a polynucleotide population can be characterized by an average relative polynucleotide length. For example, nucleic acid molecules can be isolated from samples, such as cells or other biological sources, and fragmented by any of a variety of methods during sample preparation. By adjusting parameters used in the fragmentation method, such as sonication time, polynucleotides of various lengths can be generated. Polynucleotides of the desired length can then be isolated from the resulting fragments. A population can be defined by the length of polynucleotides that are characteristic of the population as a whole, even though the lengths of individual polynucleotides of the population can be different from the polynucleotide lengths of the population as determined. In another example, a polynucleotide length characteristic of a population can be predetermined by polymerizing polynucleotides of predetermined length from a designed template.

The population of polynucleotides can have the following lengths: about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides , about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,050 nucleotides, about 1,200 nucleotides, about 1,250 nucleotides, about 1,300 nucleotides, about 1,350 nucleotides, about 1,400 nucleotides, about 1,450 nucleotides, about 1,500 nucleotides, about 1,550 nucleotides, about 1,600 nucleotides, about 1,650 nucleotides, about 1,700 nucleotides, about 1,750 nucleotides, about 1,800 nucleotides, about 1,850 nucleotides, about 1,900 nucleotides Nucleotides, about 1,950 nucleotides, about 2000 nucleotides or longer.

Features may also include other aspects of population preparation, such as nucleotide populations (DNA libraries) prepared by different library preparation methods or library preparation kits from different suppliers. The effects of aspects of the clustering process can also be compared to determine whether and how such conditions affect biases or assumptions associated with features. Each of two or more populations of polynucleotides that are distinguished from each other by one or more characteristics can undergo one, two or more copying processes, such as clustering processes, where conditions are vary between processes. Whether copy bias results from differences in traits under one condition and/or another condition or whether the amount or presence of bias differs depending on which copy condition it experiences conditions to change. In one example, multiple conditions may be modified, or several different instances of the conditions may be compared. Examples of conditions that can be modified include components of the reaction solution for solid-state copying, such as clustering processes (such as the polymerase used, pH, concentration of polynucleotide or any component, linearase used such as LMX1 or LMX2 or others, including potency additives such as GP32 or UvsX or other nucleotide binding proteins, polyethylene glycol, phosphocreatine or other additives, the type of substrate surface, or the surface on which solid phase PCR or clustering occurs type, presence or thickness of polymer coating), number or duration of copy wheels, temperature, etc. In some examples, methods of making polynucleotide populations and/or copying methods, such as any aspect during cluster formation from polynucleotide populations, can be modified and evaluated according to the methods disclosed herein to determine bias potential.

Non-limiting examples of parameters that can be altered as features in reagents used in methods used to make copies (eg, ExAmp, bridge or other clustering processes) include various enzyme concentrations (and ratios), additives (concentrations and ratios) , solution pH, polynucleotide concentration included in the solution for copying, nucleotide concentration included in the solution for copying.

The temperature at which copying occurs (eg, at, above or below about 20°C, in one example), the duration of the polymerization or wash steps, the method or duration of reagent replenishment, the introduction of reagents into fluid cells or other substrates used Non-limiting examples of flow rates, etc., can be modified and studied accordingly. Clustering time can vary as a characteristic (eg, lasts less than about 30 minutes, or within about 30 minutes to about one hour, or within about one hour to about two hours, or within about two hours to about three hours, or within within about three hours to about four hours, or within about four hours to about five hours, or within about five hours to about six hours, or within about six hours to about seven hours, or within about seven hours to about eight within hours, or within about eight hours to about nine hours, or within about nine hours to about ten hours, or within about ten hours to about eleven hours, or within about eleven hours to about twelve hours, or within about twelve hours to about twenty-four hours, or within about twenty-four hours to about thirty-six hours, or within about thirty-six hours to about forty-eight hours, or within about forty-eight hours to about seven within twelve hours, or longer). The duration of various aspects of the copying or clustering process, such as the duration of incubation of the reagent in solution, can vary as a characteristic (eg, for about 10 seconds, or about 20 seconds, or about 30 seconds, or about 40 seconds, or about 50 seconds, or about 60 seconds, or about 70 seconds, or about 80 seconds, or about 90 seconds, or about 100 seconds, or about 110 seconds, or about 120 seconds or more).

The fluid velocity or the rate at which the reagent flows through the fluid cell can vary as a characteristic (eg, the flow rate can be about 10 μl/min, or about 20 μl/min, or about 30 μl/min, or about 40 μl/min, or about 50 μl/min, or about 60 μl/min, or about 70 μl/min, or about 80 μl/min, or about 90 μl/min, or about 100 μl/min, or about 110 μl/min, or about 120 μl/min, or about 130 μl/min, or about 140 μl/min, or about 150 μl/min or higher). Other aspects that can be modified as features include pH (eg above or below or at about pH 7.5), buffer type (eg Tris based or others) and concentration of buffer or other components or cluster solution components ( For example about 100 nM, or less or more than about 100 nM).

In one example, polynucleotides from two or more populations can be combined in solution, and the solution added to a substrate such as a fluid cell for copying, including, for example, copying by a clustering process. Different identifier sequences on the polynucleotides of the two or more populations allow for the identification of populations whose surface-linked copies are copies. In one example, the relative proportion of polynucleotides from the population to the total amount of polynucleotides added can be controlled and varied in several different solutions. For example, the total number of polynucleotides in solution added to a fluid cell or lane of a fluid cell can have about equal proportions of polynucleotides from each of the two polynucleotide populations. The total number of polynucleotides in another solution added to the fluid cell or lane of the fluid cell can have about 25% polynucleotides from one polynucleotide population and about 75% polynucleotides from another polynucleotide population polynucleotides, and the total number of polynucleotides added to another solution in the fluid cell or lane of the fluid cell can have about 75% polynucleotides from another polynucleotide population and about 25% from one polynucleotide Polynucleotides of acid groups. Any other splits between the proportions of polynucleotides from one population and another can be used in different solutions (eg, about 5%/95%, about 10%/90%, about 15%/85%, about 20% /80%, about 25%/75%, about 30%/70%, about 35%/65%, about 40%/60%, about 45%/55%, about 50%/50%, about 55%/ 45%, about 60%/40%, about 65%/35%, about 70%/30%, about 75%/25%, about 80%/20%, about 85%/15%, about 90%/10 %, about 95%/5%, or any intermediate relative ratio).

library preparation

Libraries comprising polynucleotides can be prepared in any suitable manner to link oligonucleotide adaptors to target polynucleotides. As used herein, a "library" is a population of polynucleotides from a given source or sample. The library includes a plurality of target polynucleotides. As used herein, a "target polynucleotide" is a polynucleotide desired to be included in a copying process such as a clustering process. The target polynucleotide can be essentially any polynucleotide having a known or unknown sequence. It can be, for example, a fragment of genomic DNA or cDNA. The target polynucleotide can be derived from a sample of the primary polynucleotide that has been randomly fragmented. Target polynucleotides can be processed into templates suitable for amplification by placing primer sequences at the ends of each target fragment, such as identifier sequences, sequences complementary to surface-linked primers, and the like. Target polynucleotides can also be obtained from primary RNA samples by reverse transcription into cDNA.

As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a compound comprising two or more covalently bonded to each other, typically via phosphodiester bonds Molecules of nucleotide monomers. Polynucleotides typically contain more nucleotides than oligonucleotides. For purposes of illustration and not limitation, polynucleotides may be considered to contain 15, 20, 30, 40, 50, 100, 200, 300, 400, 500 or more nucleotides, and oligonucleotides may be considered Contains 100, 50, 20, 15 or fewer nucleotides.

Polynucleotides and oligonucleotides may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). These terms should be understood to include equivalently DNA or RNA analogs made from nucleotide analogs and apply to both single-stranded (such as sense or antisense) and double-stranded polynucleotides. As used herein, the term also encompasses cDNA, which is complementary or copy DNA produced from an RNA template, eg, by the action of reverse transcriptase.

Primary polynucleotide molecules can originate in double-stranded DNA (dsDNA) form (eg, genomic DNA fragments, PCR and amplification products, and the like), or can originate in single-stranded form as DNA or RNA, and converted into dsDNA form. For example, an mRNA molecule can be copied into double-stranded cDNA using standard techniques well known in the art. The precise sequence of the primary polynucleotide is generally not material for the disclosure presented herein, and may or may not be known.

In some examples, the primary target polynucleotide is an RNA molecule. In one aspect of such an example, RNA isolated from a particular sample is first converted to double-stranded DNA using techniques known in the art. Double-stranded DNA can then be indexed with library-specific tags. Different preparations of such double-stranded DNA, including library-specific index tags, can be generated in parallel from RNA isolated from different sources or samples. Subsequently, different preparations of double-stranded DNA including different library-specific index tags can be mixed with all copies, and the identity of each sequenced fragment determined with respect to the population from which it was isolated/derived by virtue of the presence of library-specific index tag sequences.

In some examples, the primary target polynucleotide is a DNA molecule. For example, a primary polynucleotide can represent the complete genetic complement of an organism, and is a genomic DNA molecule, such as a human DNA molecule, that includes intronic and exonic sequences (coding sequences), as well as non-coding regulatory sequences , such as promoter and enhancer sequences. Although it is envisaged that specific subsets of polynucleotide sequences or genomic DNA may also be used, such as specific chromosomes or portions thereof. In many instances, the sequence of the primary polynucleotide is unknown. DNA target polynucleotides can be chemically or enzymatically treated before or after a fragmentation process, such as a random fragmentation process, and before, during, or after adaptor oligonucleotide ligation.

In one example, the primary target polynucleotide is fragmented to an appropriate length for sequencing. The polynucleotide of interest can be fragmented in any suitable manner. Preferably, the target polynucleotides are randomly fragmented. Random fragmentation refers to fragmentation of polynucleotides in a disordered manner, eg, by enzymatic, chemical, or mechanical means. Any suitable fragmentation method can be used. For clarity, generating such smaller fragments via specific PCR amplification of smaller fragments of larger polynucleotides is not equivalent to fragmenting the larger polynucleotides because the larger polynucleotides remain Intact, that is, not fragmented by PCR amplification (although methods as disclosed herein can be performed on polynucleotide populations generated by either technique). In addition, random fragmentation is designed to generate fragments regardless of the sequence identity or position of the nucleotides including and/or surrounding the break.

In some examples, random fragmentation is performed by mechanical means, such as spraying or sonication, to produce a length of about 50 base pairs to about 1500 base pairs in length, such as a length of 50 to 700 base pairs or Fragments of 50 to 500 base pairs in length.

Fragmentation of polynucleotide molecules by mechanical means such as spraying, sonication, and Hydroshear can generate fragments with blunt ends and a heterogeneous mixture of 3' and 5' overhanging ends. Fragment ends can be repaired using methods or kits known in the art, such as the Lucigen DNA Terminator End Repair Kit, to generate ends that are optimal for insertion, eg, into blunt sites of a selection vector. In some examples, fragments of the nucleic acid population are blunt-ended. Fragment ends can be blunt and phosphorylated. Phosphate moieties can be introduced via enzymatic treatment, eg, using polynucleotide kinases.

In some examples, the target polynucleotide sequence is prepared using a single overhanging nucleotide by, for example, the activity of certain types of DNA polymerases, such as Taq polymerase or Klenow exo minus polymerase, which have the ability to convert a single Deoxynucleotides, such as deoxyadenosine (A), are added to, for example, the 3' end of PCR products for template-independent terminal transferase activity. Such enzymes can be used to add a single nucleotide "A" to the blunt 3' end of each strand of a polynucleotide duplex of interest. Thus, "A" can be added to the 3' end of the repaired duplex strands at each end of the target polynucleotide duplex by reaction with Taq or Klenow exo minus polymerase, and the adaptor polynucleotide construct can be T-constructs with compatible "T" pendants present on the 3' end of each duplex region of the adaptor construct. This end modification also prevents the target polynucleotide from self-ligation, so that there is a bias towards forming a combined ligated adaptor-target polynucleotide.

In some instances, fragmentation is done via tagging. In such methods, a translocase is used to fragment the double-stranded polynucleotide and ligate a universal primer sequence into one of the strands of the double-stranded polynucleotide. The resulting molecule can be gap-filled and amplified, for example, by PCR, using primers that include the 3' end with a sequence complementary to the sequence of the universal primer to which it is attached and the 5' end of the other sequence containing the adaptor to extend.

The adaptor can be attached to the target polynucleotide in any other suitable manner. In some instances, adaptors can be introduced in a single-step process. In some examples, the adaptor can be introduced in a multi-step process, such as a two-step process, which involves ligating a portion of the adaptor to a target polynucleotide having a universal primer sequence. The second step involves extension, eg, by PCR amplification, using primers that include a 3' end having a sequence complementary to the sequence of the universal primer to which it is attached and a 5' end containing additional sequences of the adaptor. Additional extensions can be performed to provide additional sequence to the 5' end of the resulting previously extended polynucleotide.

In some examples, the entire adaptor is ligated to the fragmented target polynucleotide. Preferably, the ligated adaptor comprises a double-stranded region ligated to the double-stranded target polynucleotide. Preferably, the double-stranded region is as short as possible without loss of function. In this context, "function" refers to the ability of the double-stranded region to form a stable double helix under standard reaction conditions. In some examples, standard reaction conditions refer to an enzyme-catalyzed polynucleotide ligation reaction (eg, incubation in a ligation buffer suitable for the enzyme at a temperature in the range of 4°C to 25°C) such that two strands of the adaptor are formed Reaction conditions that maintain partial adhesion during adaptor ligation to target molecules. Ligation methods utilize ligases such as DNA ligases to effect or catalyze, in this case, the ligation of the ends of the two polynucleotide strands of the adaptor duplex oligonucleotide and the target polynucleotide duplex, such that a covalent bond. The adaptor duplex oligonucleotide may contain a 5'-phosphate moiety to facilitate conjugation to the 3'-OH of the target polynucleotide. The polynucleotide of interest may contain a 5'-phosphate moiety, which is residual from the cleavage process, or added using an enzymatic treatment step, and which has been end repaired, and optionally extended by one or more overhanging bases, resulting in a suitable Joined 3'-OH. Linking in this context means covalent linkage of polynucleotide strands that were not previously covalently linked. In one aspect, such linkage is by forming a phosphodiester linkage between the two polynucleotide strands, although other covalent linkages (eg, non-phosphodiester backbone linkages) can be used.

Any suitable adaptor can be ligated to the target polynucleotide via any suitable process, such as those described above. The adaptor includes library-specific indexing tag sequences. Index tag sequences can be ligated to target polynucleotides from each library prior to sample immobilization for sequencing. The index tag itself is not formed from part of the target polynucleotide, but becomes part of the template used for amplification. The index tag can be a synthetic sequence of nucleotides added to the target as part of the template preparation step. Thus, a library-specific index tag is a nucleic acid sequence tag attached to each of the target molecules of a particular library, the presence of which indicates or is used to identify the library from which the target molecule was isolated.

Preferably, the index tag sequence is 20 nucleotides or less in length. For example, the index tag sequence can be 1 to 10 nucleotides or 4 to 6 nucleotides in length. The four nucleotide index tags give the possibility to multiplex 256 samples on the same array, and the six base index tags enable processing of 4,096 samples on the same array.

The adaptor may contain more than one index tag (or identifier sequence) so that the multiplexing potential can be increased.

An adaptor can include a double-stranded region and a region including two non-complementary single strands. The double-stranded region of the adaptor can have any suitable number of base pairs. Preferably, the double-stranded region is a short double-stranded region typically comprising 5 or more contiguous base pairs formed by bonding two partially complementary polynucleotide strands. This "double-stranded region" of an adapter refers to the region where the two strands are bonded and does not imply any particular structural configuration. In some examples, the double-stranded region includes 20 or less contiguous base pairs, such as 10 or less or 5 or less contiguous base pairs.

The stability of the double-stranded region can be increased by including non-natural nucleotides that exhibit stronger base pairing than standard Watson-Crick base pairs, and thus may be reduced in length. The two strands of the adapter are 100% complementary in the double-strand region.

When the adaptor is ligated to the target polynucleotide, the non-complementary single-stranded regions can form the 5' and 3' ends of the polynucleotide to be sequenced. The term "non-complementary single stranded region" refers to a region of an adaptor in which the two polynucleotide strands forming the adaptor exhibit sequences such that the two strands cannot be used in a PCR reaction. The degree of non-complementarity that fully adhere to each other under standard adhesion conditions.

Non-complementary single-stranded regions are provided by different portions of the same two polynucleotide strands that form the double-stranded region. The lower limit on the length of the single-stranded portion will typically be determined by, for example, the function of providing suitable sequences for binding of primers for primer extension, PCR and/or sequencing. In theory, there is no upper limit to the length of the mismatched region, except that in general it is advantageous to minimize the overall length of the adaptor, for example, in order to facilitate separation of unbound adaptor from the adaptor after one or more ligation steps Splicer-target construct. Thus, it is generally preferred that the non-complementary single-stranded region of the adaptor is 50 or less contiguous nucleotides in length, such as 40 or less, 30 or less, or 25 or less in length Fewer consecutive nucleotides.

The library-specific index tag sequence can be located in the single-stranded region, the double-stranded region, or spanning the single-stranded and double-stranded regions of the adaptor. Preferably, the index tag sequence is in the single-stranded region of the adaptor.

In addition to the index tag sequence, the adaptor may include any other suitable sequence. For example, an adaptor can include a universal extension primer sequence, which is typically located at the 5' or 3' end of the adaptor and the resulting polynucleotide used for sequencing. Universal extension primer sequences can hybridize to complementary primers bound to the surface of the solid substrate. Complementary primers include the free 3' end from which a polymerase or other suitable enzyme can add nucleotides to extend the sequence using the hybrid library polynucleotide as a template such that the reverse strand of the library polynucleotide is coupled to the solid surface. Such extension can be part of a sequencing operation or cluster amplification.

In some examples, the adaptor includes one or more universal sequencing primer sequences. A universal sequencing primer sequence can be combined with a sequencing primer to allow sequencing of an index tag sequence, a target sequence, or an index tag sequence and a target sequence.

The exact nucleotide sequence of the adaptor is generally not material and can be selected by the user such that the desired sequence element is ultimately included in the universal sequence of the library derived from the adaptor's template, e.g., for a particular set of universal extension primers and/or Or sequencing primers provide binding sites.

Adaptor oligonucleotides may contain exonuclease resistance modifications, such as phosphorothioate linkages.

Preferably, adaptors are attached to both ends of the target polypeptide to generate a polynucleotide having a first adaptor-target-second adaptor sequence of nucleotides. The first and second adaptors can be the same or different. If the first and second adaptors are different, at least one of the first and second adaptors includes a library-specific identifier sequence.

"first adapter-target-second adapter sequence" or "adapter-target-adapter" sequence means an adapter The orientation of the subunits relative to each other and the target does not necessarily mean that the sequences may not include additional sequences, such as linker sequences.

Other libraries, each comprising at least one library-specific indexing tag sequence or combination of indexing tag sequences that are different from indexing tag sequences or combinations of indexing tag sequences from other libraries, can be prepared in a similar manner.

As used herein, "attached" or "bound" are used interchangeably in the context of an adaptor relative to a target sequence. As described above, any suitable procedure can be used to ligate the adaptor to the target polynucleotide. For example, an adaptor can be ligated via ligation with a ligase; by ligating a portion of the adaptor and adding the other or remainder of the adaptor by extension, such as PCR, wherein the primer contains a combination of the other or remainder of the adaptor Incorporation of a portion of the adaptor via translocation and addition of the other or remaining portion of the adaptor via extension, such as PCR, wherein the primer contains the other or remaining portion of the adaptor; or a similar process ligated to the target. Preferably, the ligated adaptor oligonucleotide is covalently bonded to the target polynucleotide.

After the adaptor is ligated to the target polynucleotide, the resulting polynucleotide can undergo a scavenging process to enhance the adaptor-target-adapter polynucleotide by removing at least a portion of the unincorporated adaptor purity. Any suitable clearance process can be used, such as electrophoresis, size exclusion chromatography, or the like. In some examples, solid phase reverse immobilization (SPRI) paramagnetic beads can be used to separate adapter-target-adapter polynucleotides from unligated adapters. While such a process may enhance the purity of the resulting adaptor-target-adapter polynucleotides, some unligated adaptor oligonucleotides may be left behind.

Methods for amplifying immobilized adaptor-target-adapter molecules include, but are not limited to, bridge amplification and kinetic exclusion. Amplification can be performed using one or more immobilized primers. One or more immobilized primers can be a lawn on a flat surface.

As used herein, the term "solid-phase amplification" refers to being performed on or in conjunction with a solid support such that all or a portion of the amplification product is immobilized on the solid support as it is formed of any nucleic acid amplification reaction. Specifically, the term encompasses solid-phase polymerase chain reactions that are reactions analogous to standard solution-phase amplification, except that one or both of the forward and reverse amplimers are immobilized on a solid support ( polymerase chain reaction) (solid phase PCR) and solid isothermal amplification. Solid phase PCR encompasses systems such as emulsions, where one primer is anchored to the beads and the other is in free solution, and colonies are formed in a solid phase gel matrix, where one primer is anchored to the surface and one is in free solution.

In some examples, the solid support includes a patterned surface. "Patterned surface" refers to the arrangement of different regions in or on the exposed layer of a solid support. The term fluid cell "support" or "substrate" refers to a support or substrate on which surface chemistry can be added. The term "patterned substrate" refers to a support having depressions defined in the middle or thereon. The term "non-patterned substrate" refers to a substantially flat support. The substrate may also be referred to herein as a "support,""patternedsupport," or "non-patterned support." The supports can be wafers, panels, rectangular sheets, molds, or any other suitable configuration. Supports are generally rigid and insoluble in aqueous liquids. The support can be inert to the chemicals used to modify the depressions. For example, the support can be inert to chemicals used to form the polymer coating, such as attaching primers to the deposited polymer coating, and the like. Examples of suitable supports include epoxysiloxanes, glasses and modified or functionalized glasses, polyhedral oligomeric silsequioxanes (POSS) and derivatives thereof, plastics including acrylic polymers, polyhedral Styrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutene, polyurethane, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefins/cyclic olefin polymers (cyclo-olefin polymer, COP) (such as ZEONOR® from Zeon), polyimide, etc.), nylon, ceramic/ceramic oxides, silica, fused silica or silica-based materials, silicon Aluminum oxide, silicon and modified silicon (such as boron-doped p+ silicon), silicon nitride (Si ₃ N ₄ ), silicon _{oxide (SiO 2} ), tantalum pentoxide (TaO ₅ ) or other tantalum oxides (TaO _x ) , hafnium oxide (HaO ₂ ), carbon, metal, inorganic glass or the like. The support can also be glass or silicon or a silicon-based polymer, such as a POSS material, optionally with a coating of tantalum oxide or another ceramic oxide on the surface. The POSS material may be the material disclosed in Kejagoas et al., Microelectronic Engineering 86 (2009) 776-668, which is incorporated herein by reference in its entirety.

In one example, the recesses can be holes, such that the patterned substrate includes an array of holes in its surface. The pores can be micropores or nanopores. The size of each hole can be characterized by its volume, hole open area, depth, and/or diameter. For example, one or more of the regions can be the portion where one or more amplification primers are present. Portions can be separated by gap regions in which no amplification primers are present. In some examples, the pattern may be a feature of columns and rows in an x-y format. In some examples, the pattern may be a repeating arrangement of portions and/or interstitial regions. In some examples, the pattern may be randomly arranged portions and/or interstitial regions.

In some examples, the solid support includes an array of holes or depressions in the surface. This can be fabricated using a variety of techniques including, but not limited to, photolithography, stamping techniques, molding techniques, and micro-etching techniques. The technique used may depend on the composition and shape of the array substrate.

Features in the patterned surface can be covalently linked gels with patterning, such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (poly(N- -(5-azidoacetamidylpentyl)acrylamide-co-acrylamide), PAZAM) holes in glass, silicon, plastic or other suitable hole arrays on solid supports (eg micropores or nanopores). This process produces a gel pad for sequencing that is stable during sequencing operations over multiple cycles. The covalent attachment of the polymer to the pores helps maintain the gel in the structured features over the lifetime of the structured substrate during multiple uses. However, in many instances the gel need not be covalently attached to the pores. For example, under some conditions, a silane-free acrylamide that is not covalently attached to any portion of the structured substrate can be used as a gel material.

In some examples, structured substrates can be fabricated by patterning a solid support material with pores (eg, micropores or nanopores); with gel materials (eg, PAZAM, SFA, or chemically modified variants thereof) , such as the azide form of SFA (azido-SFA)) coating patterned supports; and polishing the gel-coated supports, such as via chemical or mechanical polishing, thereby retaining the gel in the pores, but Substantially all of the gel will be removed or inactivated from the interstitial regions on the surface of the structured substrate between the wells. The primer nucleic acid can be attached to the gel material. A solution of target nucleic acid (eg, a fragmented human genome) can then be contacted with the polished substrate so that individual target nucleic acids can seed individual wells via interaction with primers attached to the gel material; however, due to the Absent or not activated, the target nucleic acid will not occupy the interstitial region. Amplification of the target nucleic acid will be limited to the pores because the absence or inactivation of the gel in the gap region prevents the outward migration of the growing nucleic acid population. The process is tunable and can be easily fabricated using conventional micro or nano fabrication methods.

The disclosed subject matter includes, as an example, "solid-phase" amplification methods in which only one amplification primer is immobilized (the other primer is present, eg, in free solution), in other examples, a solid support can be Has immobilized forward and reverse primers. Some examples include "plurality" of the same forward primer and/or "plurality" of the same reverse primer immobilized on a solid support, as the amplification process may include excess primers to maintain amplification. References herein to forward and reverse primers should therefore be construed to encompass "plurality" of such primers unless context dictates otherwise.

Any given amplification reaction includes at least one type of forward primer and at least one type of reverse primer specific for the template to be amplified. However, in certain examples, the forward and reverse primers can include template-specific portions having the same sequence, and can have the exact same nucleotide sequence and structure (including any non-nucleotide modifications). In other words, it is possible to use only one type of primer for solid-phase amplification, and such single-primer methods are encompassed within the scope of the present invention. Other examples may use forward and reverse primers that contain the same template-specific sequence but differ in some other structural feature. For example, one type of primer can contain non-nucleotide modifications that are not present in another type of primer.

The terms "cluster" and "colony" are used interchangeably herein to refer to discrete sites on a solid support comprising a plurality of identical immobilized nucleic acid strands and a plurality of identical immobilized complementary nucleic acids share. The term "clustered array" refers to an array formed from such clusters or colonies. In this context, the term "array" should not be understood as requiring an ordered arrangement of clusters.

The terms "solid phase" or "surface" are used to mean a flat array in which the primers are attached to a flat surface, such as a glass, silica or plastic microscope slide or similar fluid cell device; beads, one of which Either two primers are attached to the beads and the beads are amplified; or an array of beads on the surface after the beads have been amplified.

Cluster arrays can be prepared using a process of thermal cycling or maintaining a constant temperature, and using changes in reagents to perform cycles of extension and denaturation. In one example, an isothermal process may advantageously include the use of low temperatures.

It will be appreciated that any of the amplification methods described herein or generally known in the art can be used with universal or target specific primers to amplify immobilized DNA fragments. Methods suitable for amplification include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), and nucleic acid sequence-based amplification ( nucleic acid sequence based amplification, NASBA). The above amplification methods can be used to amplify one or more related nucleic acids. For example, PCR including multiplex PCR, SDA, TMA, NASBA, and similar methods can be used to amplify immobilized DNA fragments. In some examples, primers specific for the polynucleotide of interest are included in the amplification reaction.

Other methods suitable for amplifying polynucleotides may include oligonucleotide extension and ligation, rolling circle amplification (RCA) or oligonucleotide ligation assay (OLA) techniques. It will be appreciated that such amplification methods can be designed to amplify immobilized DNA fragments. For example, in some examples, amplification methods can include ligation probe amplification or oligonucleotide ligation assay (OLA) reactions that contain primers specific for the nucleic acid of interest. In some examples, amplification methods can include primer extension-ligation reactions containing primers specific for the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that can be specifically designed to amplify a nucleic acid of interest, amplification can include primers for GoldenGate assays (Illumina, Inc., San Diego, CA).

Exemplary isothermal amplification methods that can be used in the methods of the invention include, but are not limited to, Multiple Displacement Amplification (MDA) or isothermal strand displacement nucleic acid amplification. Other non-PCR-based methods that can be used in the present invention include, for example, strand displacement amplification (SDA) or hyperbranched strand displacement amplification. Isothermal amplification methods can be used with strand displacement Phi 29 polymerase or Bst DNA polymerase large fragments, 5'->3' exo- for random primer amplification of genomic DNA. Use of these polymerases takes advantage of their high persistence and strand displacement activity. The high sustaining force allows the polymerase to generate fragments of 10 to 20 kb in length. As described above, smaller fragments can be generated under isothermal conditions using polymerases with low sustaining force and strand displacement activity, such as Klenow polymerase.

DNA polymerases can include polymerases that have been classified by structural homology into families identified as A, B, C, D, X, Y, and RT. DNA polymerases in family A include, for example, T7 DNA polymerase, eukaryotic mitochondrial DNA polymerase gamma, E. coli DNA Pol I (including Klenow fragments), Thermus aquaticus Pol I and Bacillus stearothermophilus Pol I. Family B DNA polymerase of the eukaryotic DNA polymerases include, for example, a, 6, and E; DNA polymerase C; (. Thermococcus sp) T4 DNA polymerase, the Phi29 DNA polymerase, thermophilic Lactococcus 9 ⁰ N-7 ancient archaeon polymerase (also known as 9°N™) and variants thereof, such as the examples disclosed in US Patent Application Publication No. 2016/0032377 A1, and RB69 phage DNA polymerase. Family C includes, for example, the E. coli DNA polymerase III alpha subunit. Family D includes, for example, polymerases derived from the Euryarchaeota subdomain of Archaea. DNA polymerases in family X include, for example, the eukaryotic polymerases Pol β, Pol σ, Pol λ and Pol μ and S. cerevisiae Pol4. DNA polymerases in family Y include, for example, Pol n, Pol 1, Pol κ, Escherichia coli Pol IV (DINB), and Escherichia coli Pol V (UmuD'2C). The reverse transcriptase (RT) family of DNA polymerases includes, for example, retroviral reverse transcriptases and eukaryotic telomerases. Example RNA polymerases include, but are not limited to, viral RNA polymerases, such as T7 RNA polymerase; eukaryotic RNA polymerases, such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V ; and Archaeal RNA polymerase. Other polymerases are also included among the polymerases as mentioned herein, as are any other functional polymerases, including those with any of the above-mentioned polymerases provided by way of non-limiting example only Polymerases compared to modified sequences.

In some examples, isothermal amplification can be performed using kinetic exclusion amplification (KEA), also known as exclusion amplification (ExAmp). Nucleic acid libraries of the invention can be made using methods comprising the steps of reacting amplification reagents to generate a plurality of amplification sites, each of the sites comprising substantial amounts of amplicons from individual target nucleic acids that have been seeded pure group. In some examples, the amplification reaction is performed until a sufficient number of amplicons are produced to fill the capacity of the respective amplification sites. Filling the capacity of the vaccinated site in this way prevents the target nucleic acid from falling at that site, thereby producing a clonal population of amplicons at that site. In some instances, apparent homogeneity can be achieved even if the amplification site does not fill capacity before the second target nucleic acid reaches the site. Under some conditions, the amplification of the first target nucleic acid can proceed to such an extent that a sufficient number of copies are made to effectively outweigh or completely override the production of copies of the second target nucleic acid transferred to the site. For example, in the example using a bridge amplification process for circular features less than 500 nm in diameter, it has been determined that after 14 cycles of exponential amplification of the first target nucleic acid, the same locus from the second target nucleic acid The number of contaminating amplicons produced by the contamination would be insufficient to adversely affect sequencing-by-synthesis analysis on the Illumina sequencing platform.

In some instances, kinetic exclusion occurs when a process occurs at a rate fast enough to effectively exclude another event or process from occurring. For example, nucleic acid arrays are fabricated in which the sites of the array are randomly seeded with target nucleic acid from solution, and copies of the target nucleic acid produced during the amplification process fill the capacity of each of the seeded sites. According to the kinetic exclusion method of the present invention, the inoculation and amplification processes can be performed simultaneously under conditions where the amplification rate exceeds the inoculation rate. Thus, a relatively fast copy production rate at the site where the first nucleic acid of interest has been seeded will effectively preclude seeding of the second nucleic acid at that site for amplification.

Kinetic exclusion can take advantage of a relatively slow initiation rate of amplification (eg, a slow rate of making a first copy of a target nucleic acid) relative to a relatively fast rate of making subsequent copies of a target nucleic acid (or first copy of a target nucleic acid) . In the example of the previous paragraph, because the relatively slow rate of target nucleic acid seeding (eg, relatively slow diffusion or transport) relative to the relatively fast rate at which amplification occurs to populate the locus with copies of the nucleic acid seed, occurs Power out. In another example, kinetic exclusion can occur because the formation of a first copy of a target nucleic acid seeded at a site is delayed (eg, delayed or slowly activated) relative to the relatively faster rate of production of subsequent copies that fill the site. In this example, several different target nucleic acids may be seeded at individual sites (eg, several target nucleic acids may be present at each site prior to amplification). However, the formation of the first copy of any given target nucleic acid can be activated randomly such that the average rate of formation of the first copy is relatively slow compared to the rate of production of subsequent copies. In this case, although several different target nucleic acids can be seeded at individual sites, kinetic exclusion will allow only one of those target nucleic acids to be amplified. More specifically, once the first nucleic acid of interest is activated for amplification, its copies will rapidly fill the capacity of the site, preventing the making of copies of the second nucleic acid of interest at that site.

Amplification reagents can include additional components that promote amplicon formation and in some cases increase the rate of amplicon formation. Examples are recombinases. Recombinases can facilitate amplicon formation by allowing repeated invasion/extension. More specifically, recombinases can facilitate invasion of target nucleic acid by a polymerase and extension of primers by a polymerase using the target nucleic acid as a template for amplicon formation. This process can be repeated in a chain reaction in which amplicons resulting from each round of invasion/extension serve as templates in subsequent rounds. Because no denaturation cycles (eg, via heat or chemical denaturation) are required, the process can proceed more rapidly than standard PCR. Thus, recombinase-promoted amplification can be performed isothermally. It is generally desirable to include ATP or other nucleotides (or in some cases, non-hydrolyzable analogs thereof) in amplification reagents facilitated by recombinases to facilitate amplification. Mixtures of recombinase and single stranded binding (SSB) proteins are particularly useful, since SSB further facilitates amplification. Exemplary formulations for recombinase-promoted amplification include formulations marketed by TwistDx (Cambridge, UK) as the TwistAmp kit.

Another example of a component that can be included in amplification reagents to facilitate amplicon formation, and in some cases increase the rate of amplicon formation, is a helicase. Helicases can facilitate amplicon formation by allowing a chain reaction of amplicon formation. Because no denaturation cycles (eg, via heat or chemical denaturation) are required, the process can proceed more rapidly than standard PCR. Thus, helicase-promoted amplification can be performed isothermally. Mixtures of helicase and single-stranded bound (SSB) proteins are particularly useful, as SSB further facilitates amplification. Exemplary formulations for helicase-promoted amplification include formulations marketed by Biohelix (Beverly, MA) as the IsoAmp kit.

Yet another example of a component that can be included in amplification reagents to promote amplicon formation, and in some cases increase the rate of amplicon formation, is an origin binding protein.

An advantage of the method described herein is that it provides rapid and efficient parallel detection of multiple target nucleic acids. Accordingly, the present invention provides integrated systems capable of producing and detecting nucleic acids using techniques known in the art, such as the examples above. Thus, the integrated systems of the present invention may include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, including, for example, pumps, valves, reservoirs, fluidic lines, temperature Components of controllers and the like. Fluid cells can be configured and/or used in integrated systems to detect target nucleic acids. As exemplified with respect to the fluid cell, one or more of the fluid components of the integrated system can be used for amplification methods and detection methods. One or more of the fluidic components of the integrated system can be used in the amplification methods described herein and in the delivery of sequencing reagents in sequencing methods, such as those exemplified above. As used herein, the term "flow cell" is intended to mean having a chamber (ie, a flow channel) in which a reaction can take place, inlets for delivering one or more reagents into the chamber, and removal from the chamber A container for the outlet of one or more reagents. In some examples, the chamber enables detection of a reaction or signal present in the chamber. For example, the chamber may include one or more transparent surfaces in the chamber that allow for optical detection of the array, optically labeled molecules, or the like. As used herein, a "flow channel" or "flow channel region" can be a region defined between two binding components that can selectively accept a liquid sample. In some examples, a flow channel can be defined between the patterned support and the lid, and thus can be in fluid communication with one or more recesses defined in the patterned support. In other examples, flow channels can be defined between the non-patterned support and the lid. Other examples may include dishes, trays, or wells for separating reactants, including automated fluidic systems for exchanging reagents and other reaction components. For example, multi-well plates can be used, including, for example, 96- or 384-well plates.

Alternatively, the integrated system may include separate fluidic systems for performing the amplification method and performing the detection method. Examples of integrated sequencing systems capable of generating amplified nucleic acids and also determining nucleic acid sequences include, but are not limited to, the MiSeq ^™ platform (Illumina, Inc., San Diego, CA).

Non-limiting examples of suitable primers include the P5 and/or P7 primers used on the surface of commercial fluid cells sold by Illumina, Inc. for HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™ , NOVASEQ™, GENOME ANALYZER™, ISEQ™, cBot with imaging (icBot) and other instrument platforms. And the portion of the template polynucleotide comprising the nucleotide sequence corresponding to or complementary to the first or second primer as disclosed above may have, for example, the same as P5 according to such primer sequence as used in the SBS platform as mentioned above. Primer (including the nucleotide sequence of AATGATACGGCGACCACCGAGATCTACAC), P7 primer (including the nucleotide sequence of CAAGCAGAAGACGGCATACGAGAT) or sequences corresponding to or complementary to both, or others.

As a non-limiting example, a substrate can include a substrate for use in any of the aforementioned SBS or other platforms, such as for automated clustering of labeled oligonucleotides hybridized to surface-linked polynucleotides and imaging platforms, which can also (but need not) be platforms equipped to perform the sequenced samples themselves of the SBS process. Such substrates may be fluid cells.

As used herein, the term "depression" refers to discrete concave features in a patterned support having surface openings that are fully surrounded by one or more interstitial regions of the patterned support surface. The recesses may have any of a variety of shapes at their openings in the surface, including, for example, circles, ovals, squares, polygons, stars (with any number of vertices), and the like. The cross-section of the depression obtained normal to the surface can be curvilinear, square, polygonal, hyperbolic, conical, angular, and the like. For example, the recesses can be holes. As also used herein, a "functionalized depression" refers to discrete concave features linked by primers, which in some instances are attached to the surface of the depression by a polymer such as PAZAM or similar polymers.

It is to be understood that ranges provided herein include the stated range and any value or sub-range within the stated range. As an example, the range of about 100 nm to about 1,000 nm should be interpreted to include not only the expressly recited limit values of about 100 nm to about 1,000 nm, but also individual values, such as about 708 nm, about 945.5 nm, etc., and subranges, such as about 425 nm to about 825 nm, about 550 nm to about 940 nm, and the like. Additionally, when "about" and/or "substantially" are used to describe a value, it is meant to encompass minor variations (up to +/- 10%) of the stated value. Example

The following examples are intended to illustrate specific embodiments of the present invention, but are in no way intended to limit its scope.

Example 1. Evaluation of linearity of different polynucleotide sizes (human 350 bp, 450 bp and 550 bp and bacterial 350 bp and 550 bp pools) and different GC/AT contents (bacterial pools).

Methods: Different ratios of different pools (population length, GC/AT content, human or bacterial pools) were used ^{for clustering on the HiSeq™} X fluid cell. Intensity is plotted against the proportional amount of DNA pool. Application of a linear fit and R ² calculated using JMP software. Clustering and hybridization imaging of fluorescently labeled oligonucleotide probes of identifier sequences on the icBot platform. Polynucleotides were labeled with either of two identifier sequences complementary to a fluorescently labeled oligonucleotide probe labeled with fluorescently labeled Alexa 647 (probe 1:/5Alex647N/CT ACA CAT AGA GGC ACA CTC or probe 2: /5Alex647N/CT ACA CGT ACT GAC ACA CTC, available from IDT). Solutions with the following polynucleotide concentrations from one or another population (or pool) were loaded onto 8 fluidic cell (FC) lanes: FC Group 1 Group 2 Lane 1 0% 100% Lane 2 20% 80% Lane 3 40% 60% Lane 4 50% 50% Lane 5 60% 40% Lane 6 80% 20% Lane 7 100% 0% Lane 8 50% 50%

Gain (40) and imaging exposure times (probe 1 600 ms and probe 2 600 ms to 900 ms), number of exposures (3), and probe incubation time (6 min) were used for surface-attached copies after clustering imaging.

In this example, the fluorescence intensity was used as a readout for the level of cluster expansion. It is important to see if the fluorescence intensity detected in this assay correlates with the level of cluster expansion/library input. A good/linear correlation between these two factors determines the basis for the analysis (and the data analysis method for this analysis).

result

350 bp human libraries: Probe 1 and Probe 2 has a similar linear, wherein R ^{2 is} about 0.99 input display library (clusters amplification) and the linear relationship between signal intensity. Data from probe 2 is shown in FIG. 3 . Similar linear relationships were found using the proportions of polynucleotides from GC-rich (eg, Rhodobacter), GC-poor (eg, Bacteroides cactus), or longer (550 bp) populations, indicating that the concentration of starting material shifted after clustering is the signal strength.

Example 2: Analytical sensitivity to determine what percentage of DNA amplification differences can be detected.

Methods: DNA inputs with 10% variance were ^{clustered on a HiSeq™} X fluidic cell. Signal intensities were measured after probe 1 or probe 2 hybridization.

result

Data from the 13 fluid cells of the 350 bp cluster of Rhodobacter are summarized in Figure 4. The JMP analysis showed that 40%, 50% and 60% of the DNA pool input of the Rhodobacter 350 bp pool could be separated by this analysis with statistical significance (95%). Similar assay sensitivities were found using libraries with different insert sizes or GC content, such as human 350 bp, 450 bp and 550 bp, Rhodobacter 550 bp and Cactobacillus 350 bp and 550 bp libraries.

It should be understood that all combinations of the foregoing concepts and additional concepts discussed in greater detail herein (provided that such concepts are not mutually incompatible) are considered part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the conclusion of this disclosure are considered part of the inventive subject matter disclosed herein, and can be utilized to achieve the benefits and advantages described herein.

none

These and other features, aspects and advantages of the present invention will become better understood when reading the following embodiments with reference to the accompanying drawings, wherein:

[FIG. 1] A flowchart showing an aspect according to one example of a method as disclosed herein.

[FIG. 2] An illustration showing elements of one example of a method according to an aspect of the present invention.

[FIG. 3] is shown in one example, according to aspects of the present invention, detected from fluorescently labeled oligonucleotides hybridized to copies of polynucleotides starting with DNA loaded at different ratios Schema of the difference in mean intensity.

[FIG. 4] is a graph comparing the fluorescence detection intensities after clustering of polynucleotides loaded at 40%, 50% or 60% of the total DNA in a clustering procedure in one example.

[FIG. 5] A flowchart showing an aspect according to one example of a method as disclosed herein.

Claims (22)

A method that includes: making two or more copies of a polynucleotide population comprising an identifier sequence, wherein the copies are attached to a substrate, hybridizing oligonucleotides to such identifier sequences, and comparing the amount of oligonucleotides hybridized to copies of the two or more polynucleotide populations, wherein At least one characteristic differs between the two or more populations of polynucleotides or between the production of copies of the two or more populations of polynucleotides attached to the substrate. The method of claim 1, wherein the at least one characteristic is selected from the group consisting of length, guanine-cytosine content and preparation method. The method of claim 1 or 2, wherein the at least one characteristic comprises guanine-cytosine content. The method of any one of claims 1 to 3, wherein the at least one characteristic comprises length. The method of any one of claims 1 to 4, wherein the at least one feature comprises a method of preparation. The method of any one of claims 1 to 5, wherein at least one characteristic differs between the production of copies of the two or more populations of polynucleotides attached to the substrate. The method of any one of claims 1 to 6, wherein the oligonucleotides comprise fluorophores. The method of any one of claims 2 to 7, further comprising detecting a difference between the amount of oligonucleotides hybridized to copies of the two or more populations of polynucleotides attached to the substrate difference, wherein the difference is at least about 10%. The method of claim 8, wherein the difference is at least about 20%. The method of claim 8, wherein the difference is at least about 30%. As in the method of claim 1, wherein: The at least one characteristic comprises a combination, and the combination comprises two or more of guanine-cytosine content, length, method of preparation, and manufacture of copies of the two or more populations of polynucleotides attached to the substrate many, the two or more polynucleotide populations comprise three or more polynucleotide populations, and The combination of each of the three or more polynucleotide populations is different from the combination of the other polynucleotide populations. The method of claim 11, further comprising detecting an amount of oligonucleotides hybridized to copies of two or more of the three or more polynucleotide populations attached to the substrate difference, wherein the difference is at least about 10%. The method of claim 12, wherein the difference is at least about 20%. The method of claim 12, wherein the difference is at least about 30%. A method that includes: making two or more copies of a polynucleotide population comprising an identifier sequence, wherein the copies are attached to a substrate, hybridizing an oligonucleotide comprising a fluorophore to the identifier sequences, and detecting the amount of oligonucleotides that hybridize to copies of the two or more polynucleotide populations, wherein at least one characteristic differs between the two or more populations of polynucleotides or between the production of copies of the two or more populations of polynucleotides attached to the substrate, and The at least one characteristic is selected from the group consisting of length, guanine-cytosine content, method of preparation, and production of copies of the two or more populations of polynucleotides attached to the substrate. The method of claim 15, wherein the at least one characteristic comprises guanine-cytosine content. A method as in any of claims 15 or 16, wherein the at least one characteristic comprises length. The method of any one of claims 15 to 17, wherein the at least one feature comprises a method of preparation. The method of any one of claims 15 to 18, wherein at least one characteristic differs between the production of copies of the two or more populations of polynucleotides attached to the substrate. The method of any one of claims 15 to 19, further comprising detecting a difference between the amounts of oligonucleotides that are heterozygous to copies of the two or more polynucleotide populations, wherein the difference to at least about 10%. The method of claim 20, wherein the difference is at least about 20%. The method of claim 20, wherein the difference is at least about 30%.

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